Physiological processes (e.g., hypoxia, acidity and changes in temperature), which are present in 90% of tumor microenvironments, are considered promising environmental markers for tumor targeting. We propose to employ intrinsic tumor acidity for the detection of tumors by non-invasive nuclear imaging. Our method is based on the pH- selective interaction of pHLIP (pH (Low) Insertion Peptide) with cell membranes. The pHLIP peptide contains 37 residues and at neutral pH it interacts weakly with the surface of membranes, but at acidic pH (<7.0) it inserts across the membrane and forms a stable transmembrane 1-helix. pHLIP conjugated to a PET or SPECT radionuclide, offers the possibility of developing a non-invasive imaging method that will be translatable to the clinical for the diagnosis of solid tumors and metastasis. The major objective of this project is to develop a new approach for the imaging of solid tumors based on pHLIP- based radiopharmaceuticals, with the final goal of translating an agent to the clinic. There are four Specific Aims we plan to undertake in the proposed study (1) To develop conjugation and labeling protocols for determining the optimal 64Cu-pHLIP-based radiopharmaceutical and to perform comparative testing in vitro and in vivo in the LnCAP and PC-3 tumor models; (2) To determine which radiometal results in the optimal in vivo tumor delivery of pHLIP; (3) To develop a conjugation protocol for the preparation of pHLIP-S-S-metal-chelate, and, secondly, to evaluate the intracellular delivery of fluorescent Eu-DOTA by pHLIP on liposomes, cell culture and in vivo by whole-body fluorescent imaging, and, (4) The most promising constructs from SA1, SA2 & SA3 will be studied with PET imaging and pharmacokinetics studies with direct correlation to tumor pHe determined by MRS studies. By combining the knowledge and experience of the two centers (URI and MSKCC), we aim to develop an optimized, novel radiometal- pHLIP peptide construct as a PET imaging agent which will be translatable to the clinic for the diagnosis and monitoring of therapy of primary cancers and metastasis in their earliest stages of development.